Molecular interactions are central to the understanding of basic biology and cellular function, are paramount to all molecular assays (research and in vitro diagnostics), are essential in biomarker discovery and critical toward evaluating therapeutics. However, the tools available to quantify these interactions have limitations, in so far as they either rely upon altering the native state of the interacting molecules by incorporating labeling schemes, demanding surface immobilization of one of the interacting moieties or require prohibitive quantities of sample. The ability to perform pure liquid-phase molecular binding analysis at high sensitivity, in a miniaturized format, is therefore desirable and has now been demonstrated with back-scattering interferometry (BSI). BSI employs a simple and inexpensive optical train comprised of a He-Ne laser, a microfluidic channel, and a position sensor monitoring minute refractive index changes. These measurements may be made within a microfluidic channel formed in glass, fused silica, or plastic and at physiologically relevant concentrations in complex matrices. Yet the current embodiment of BSI is limited. Tedious alignment methods, immature transduction schemes, poorly refined sample handling and introduction methods, expensive disposable chips, and performance limitations due to environmental noise sensitivity, all impede the wide dissemination and adoption of BSI in the life science and drug discovery communities. We propose to refine BSI, facilitating commercialization by Molecular Sensing Inc. and allowing the subsequent broad dissemination in the biological research community (under a future Phase II grant). Under this Phase I project, we will enable transfer to the industrial sector by performing three specific aims: 1) constructing an optically simple two channel BSI for enhanced S/N and environmental noise compensation, 2) simplifying alignment and improving performance through the use of a cross-correlation based position sensing algorithm and 3) implementing a user friendly graphical interface (GUI). Model interaction systems will be used to demonstrate that BSI gives meaningful and quantitative binding affinity values (from <M to pM) and that it can be used to screen for molecular interactions in various matrices (serum, cell-free media, DMSO). PUBLIC HEALTH RELEVANCE: Proteins interact constantly with one another and with other entities in the cellular and extracellular environment. However, sensitive in vitro methods to monitor such interactions, in particular methods that are label-free and do not involve surface immobilization, have until recently been lacking. Free-solution, label-free, molecular interactions can now be investigated using back-scattering interferometry (BSI), allowing quantification of KD values ranging from micromolar to picomolar using miniscule quantities of the binding pairs. With BSI, little a priori knowledge about the proteins, antibodies, or drug targets etc. is necessary to quantify the level of interaction and screen for efficacy or perform a diagnosis. The BSI methodology has the potential to shift the paradigm for molecular interaction studies, allowing screening for unknown binding partners of orphan receptors, study of inhibitors for GPCR targets, as well as for numerous other binding pairs and is likely applicable to diagnostics and therapeutic monitoring in human specimens. Furthermore, the technology is simple and inexpensive allowing for the potential of BSI to be widely disseminated.